Feline leukemia virus, a horizontally transmitted retrovirus, was first discovered among cats living in an urban environment and having frequent social contact (Jarrett, et al., 1964, Nature 202: 566-567). FeLV is believed to have been the first naturally occurring retrovirus in which contagious spreading was documented (Jarrett, et al., 1964, Nature 202: 566-567; Kawakami, et al., 1967, Science 158: 1049-1050). The FeLV retrovirus can cause either proliferative (lymphosarcoma) or antiproliferative diseases (aplastic anemia and immunodeficiency syndrome) in cats (Anderson, et al., 1971, J. Natl. Cancer Inst. 47: 807-817; Hardy, et al., 1976, Cancer Res. 36: 582-588). Regardless of the clinical course of infection, however, the FeLV establishes a permanent infection in the cat that is not eliminated. Therefore, prophylactic vaccines are critical to prevent FeLV infection in cats.
A viral vaccine should be designed such that both a humoral and CTL response is achieved, as well as stimulating a useful immunological memory. Examples of potential vaccines against viral infection include attenuated or inactivated whole virus, purified viral macromolecules (such as envelope proteins or capsular polysaccharides), recombinant antigen vaccines, recombinant vector vaccines and synthetic subunit vaccines.
The most common type of viral vaccine developed to date have been whole organism vaccines that are either attenuated or inactivated. An attenuated viral vaccine is a virus that has lost its pathogenicity, but not its ability for transient growth within the host. Major advantages of the attenuated viral vaccine include prolonged exposure to the immune system due its ability for transient growth as well as its ability to induce both a humoral and CTL response. Since an attenuated viral vaccine is a mutant, avirulent organism selected from culturing of a wild type, virulent organism, the possibility exists that the attenuated viral vaccine could revert to a virulent form subsequent to host vaccination. An inactivated viral vaccine is usually produced by exposing a virulent strain to either chemical or radiation treatment. Such inactivated strains cannot, as a rule, revert to a virulent strain. However, such vaccines tend to induce only a humoral immune response and usually require multiple boosters due to an inability to grow transiently within the host.
Purified macromolecules utilized as a vaccine reduce the risk of reversion to a virulent form. However, utilizing a macromolecule such as an envelope protein or capsular polysaccharide most likely results only in an induction of a humoral response. A CTL response may be induced by proper selection and presentation of an immunizing antigen.
Viral antigen vaccines can be expressed and purified utilizing recombinant DNA techniques. A DNA sequence encoding an antigen determinant is isolated, characterized and subcloned into an appropriate DNA expression vector. The expression vector, (usually plasmid DNA) is transformed into an appropriate host (e.g., E. coli, yeast or a mammalian cell line), grown under conditions amenable for expression of the cloned antigen determinant, and purified for use as a vaccine. The recombinant proteins or peptides are usually processed as an exogenous antigen, more often resulting in a humoral but not a CTL response.
A genetically engineered virus can be utilized as a vector. The viral vector contains a recombinant DNA sequence encoding an antigen determinant and is subcloned downstream of a viral vector promoter. The recombinant DNA sequence (again, most likely plasmid DNA) is transferred into the viral vector genome such that the DNA sequence encoding the antigenic determinant is expressed. The recombinant viral vector is then administered, for example, by dermal scratching. A localized infection ensues, allowing the antigen determinant to be expressed, inducing both a humoral and cellular response within the vaccinated host. For a review of these hereinbefore described methods available to the skilled artisan, see Kuby, 1992, In: Immunology; Chapter 18, "Vaccines"; W. H. Freeman, New York, N.Y.
Olsen, et al. (1977, Cancer Res., 37: 2082-2085) immunized cats with a combined vaccine composed of a killed FeLV virus and killed feline oncornavirus-associated cell membrane antigen (FOCMA) tumor cells. The combined vaccine did not inhibit the induction of FeLV viremia. (Also, see Pederson, et al., 1979, Am. J. Vet. Res. 40: 1120-1126, which exemplifies the difficulty in generating immunity via vaccination with killed FeLV virus.)
Hoover, et al., (1991, J. Am. Vet. Med. Assoc. 199: 1392-1401) tested inactivated FeLV virus, live FeLV virus and FeLV envelope peptide prototypes as potential vaccines. An inactivated vaccine developed from the FeLV-FAIDS-61E-A isolate protected cats from both homologous and heterologous viral exposure. In contrast, a panel of FeLV-GA-B envelope peptides, including peptides representing portions of the immunodominant domain, major neutralizing domain and variable neutralizing domain of gp70, were unsuccessful in providing resistance against FeLV challenge.
Nicolaisen-Strouss (1987, J. Virol. 61: 3410-3415) identified an FeLV variant that was not neutralized by the 5-amino acid epitope described below by Elder, et al. A single amino acid change (proline to leucine) three amino acids from the NH.sub.2 -terminus of the 5-amino acid binding epitope was implicated in lowering the affinity for binding the neutralizing antibody.
The FeLV envelope protein, gp70, was substantially purified and used to vaccinate cats (Pederson, et al., 1986, Vet. Immunol. Immunopathol. 11: 123-148). Although serum antibodies were produced, no viral neutralizing antibodies were detected in vaccinated cats. Additionally, gp70 vaccinated cats became more persistently retroviremic than non-immunized cats subsequent to a virulent FeLV challenge.
Gilbert, et al., 1987, Virus Res. 7: 49-67) disclosed a lack of FeLV neutralizing activity or immunoprophylaxis following immunization with a vaccinia vector expressing the env gp85 protein of FeLV-GA-B.
Subunit vaccines to combat FeLV infection were developed utilizing FeLV antigens released from lymphoid cells persistently infected with the Kawakami isolate (Lewis, et al., 1981, Infect. Immun. 34: 888-894; Mastro, et al., 1986, Vet. Immunol. Immonopath. 11: 205-213; reviewed in Lewis, et al., 1988, Vet. Microbiology 17: 297-308). This cell line produces viral coded polypeptides which are subsequently harvested and used to vaccinate cats. These viral antigens compose a non-infectious subunit vaccine which is approximately 80% effective in preventing FeLV viremia.
Subunit vaccines may be designed in attempts to structurally mimic domains of a viral envelope protein, in part due to the fundamental role these proteins play in virus entry into the host cell and subsequent cytopathic effects (Kowalski, et al., 1987, Science 237: 1351-1355). For example, the retrovirus external surface unit (SU) protein is involved in binding to a host cell receptor, followed by a fusion event mediated by the transmembrane (TM) protein (Dalgleish, et al., 1984, Nature 312: 763-766; Bosch, et al., 1989, Science 244: 694-697; Battini, et al., 1992, J. Virol. 66: 1468-1475). Therefore, these proteins may be targeted in immunological strategies for prophylactic or therapeutic treatment of a viral infection.
Absent detailed structural characterization by techniques such as NMR spectroscopy or crystallography, the structure and function of these envelope proteins has been investigated through indirect means. For example, inferences concerning structural and functional domains of retroviral envelope proteins have been investigated by utilizing (1) chimeric viruses (Donahue, et al., 1991, J. Virol. 65: 4461-4469; Battini, et al., 1992, J. Virol. 66: 1468-1475), (2) site-directed mutagenesis (Bosch, et al., 1989, Science 244: 694-697; Willey, 1989, J. Virol. 63: 3595-3600), and (3) small synthetic peptides and specific antibodies (Palker, et al., 1988, Proc. Natl. Acad. Sci. USA 85: 1932-1936; Dyson, et al., 1992, Biochemistry 31: 1458-1463; Nick, et al., 1990, J. Gen. Virol. 71: 77-83).
Nunberg, et al. (1990, Proc. Natl. Acad. Sci. USA 81: 3675-3679) disclosed a method to map antigenic determinants of a specified protein. DNA fragments are generated by DNaseI digestion and subcloned into the .beta.-galactosidase gene of the lambda phage Charon 16. Random peptides representing the coding region of the entire protein are expressed and screened with a specific monoclonal antibody to determine its epitope. Binding of a gp70 neutralizing antibody was mapped to a 14 amino acid region (213-226) of the gp70 protein.
Elder, et al. (1987, J. Virol. 61: 8-15) synthesized short peptides corresponding to regions of FeLV envelope proteins, gp70 and p15E. These peptides were conjugated to keyhole limpit hemocyanin and injected into rabbits to induce anti-peptide antibody production. The sera was then tested for the ability to neutralize FeLV isolates. A five amino acid sequence within the gp70 coding region was determined to be required for neutralizing activity. The five amino acid epitope was the core of a number of short peptides to which antisera had been raised. The longest of these core peptides was 18 amino acids.
Nick, et al. (1990, J. Gen. Virol. 71: 77-83) synthesized 19 peptides, from 7-19 amino acid residues in length. These peptides were conjugated to keyhole limpit hemocyanin and the anti-peptide sera was tested for neutralizing ability against FeLV. One gp70 anti-peptide antibody involved in FeLV neutralization was raised against the peptide representing amino acids 221-227. This epitope is contained within a portion of the neutralization domain described by Nunberg, et al. (1990, Proc. Natl. Acad. Sci. USA 81: 3675-3679). However, anti-peptide antibodies raised against a peptide representing amino acids 228-234 of gp70, also contained within the Nunberg neutralization domain, did not neutralize FeLV in this study. The clustering of neutralization sites is located within a proline-rich sequence located between amino acids 230-289 of gp70 (Stewart, et. al., 1986, J. Virol. 58: 825-834; Donahue, et al., 1988 J. Virol. 62: 722-731).
A peptide vaccine providing initial protection against an FeLV challenge or as a booster to an initial vaccination against FeLV would be of immense value. A humoral response, cellular response and stimulation of an immunological memory would most likely result from a peptide retaining a secondary structure corresponding to the equivalent amino acid domain of an in vivo protein.